Sulphate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine, preparation thereof and use of the same

ABSTRACT

The present invention relates to sulphate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine and pharmaceutically acceptable solvates thereof, preparation thereof, pharmaceutical compositions containing them and use of the same in the treatment and/or prevention of neurodegenerative diseases.

This application is a divisional of U.S. patent application Ser. No.14/655,642, filed Jun. 25, 2015, which is a U.S. national phase ofInternational Application No. PCT/EP2013/078068, filed Dec. 27, 2013,which claims priority from European Patent application no. EP 12306690,filed Dec. 27, 2012, the disclosure of each of which is herebyincorporated by reference in its entirety.

The present invention relates to novel sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineand pharmaceutically acceptable solvates thereof, their preparation,pharmaceutical compositions containing them and use of the same in thetreatment and/or prevention of neurodegenerative diseases.

BACKGROUND OF THE INVENTION

N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-aminewhich has the structure of Formula I

belongs to a family of 1,4-bis(3-aminopropyl)piperazine derivativespreviously disclosed in WO2006/051489 and which are useful for thetreatment and/or prevention of neurodegenerative diseases.

The free base form of this compound presents long term stability issueslikely due to oxidations occurring at the piperazine ring. In addition,the free base form does not crystallize. In terms of pharmaceuticaldevelopment, crystalline forms of active ingredients are indeedpreferred. They generally overcome stability issues and open up tocrystallization and/or recrystallization processes which are suitablefor industrial scale purification, high batch to batch reproducibility,in particular with regards to crystallinity.

Although it is known that the salification of a pharmaceutically activemolecule (PAM) may improve its physico-chemical properties, theselection of a suitable salt remains a complex process. Indeed,improving physico-chemical properties goes way beyond obtaining stablesolid materials. These solids must comprise a crystallized phase whichhas a good crystallinity and a defined morphology. In other aspects,salt forms may provide other benefits such as improving water solubilitybut can also be equally detrimental due to hygroscopicity, stabilityissues or intreseque toxicity. Hence, salt selection cannot be madearbitrarily and warrants in depth studies in the first place.

The oxalate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,which by the way was reported in WO2006/051489, partly overcomes theabove-mentioned drawbacks encountered with the free base. However, 4equivalents of oxalic acid are necessary to obtain the oxalate salt as astable powder. Consequently, the percentage by weight ofpharmaceutically active molecule with oxalate salts drops to as low as54%. The amount of drug to be administered for a given dose of PAM isthus significantly increased.

Furthermore, oxalates are not considered as one of the mostpharmaceutically acceptable salt anymore. Oxalate salts are in certaincases nephrotoxic, for instance naftidofuryl oxalate is known to causecalcium oxalate crystalluria and thus kidney stones in elderly patients.In addition, due to the low solubility of calcium oxalate, increasedconcentration of calcium oxalate in body fluids, including urine(hyperoxaluria), can lead to the deposition of calcium oxalate(oxalosis) in the kidney tissue (nephrocalcinosis) or urinary tract(urolithiasis). Oxalosis can involve many different organs when kidneysfail to clear calcium oxalate. Deposits in blood vessels can causepainful skin ulcers that do not heal, deposits in bone marrow causeanaemia and deposits in the heart cause abnormalities of heart rhythm orpoor heart function. Patients suffering from neurodegenerative diseasesare generally old and suffer from other pathologies for which theyreceive other medications. Their kidneys are thus already highlysolicited for the excretion of the drugs taken by these patients who bythe way are less aware of thirst sensations and thus prone todehydration. Excess calcium oxalate is eventually excreted in patientswho have healthy kidneys and who drink a lot of water. Excess calciumoxalate is indeed a real issue in therapy, especially for the elderly.In addition, treatments of neurodegenerative diseases are often chronic,over very long periods of time, if not over lifetime. Applicantconsidered that due to their potential kidney and urinary tracttoxicity, oxalate salts would add an undue burden to patients alreadyweakened by their condition. Applicant thus considered oxalate salts aspharmaceutically unacceptable, especially sinceN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-aminerequires 4 equivalents of the salt counter-ion to remain stable.Therefore, even though oxalate salts allow obtaining stable solidmaterials, they imply risking toxic side effects.

There is thus still a need in the art for stable, crystalline and nonhygroscopic salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-aminethat do not present the above-mentioned drawbacks in terms of saltformation and toxicity.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected findings that sulphatesalts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineprovide stable free flowing crystalline powders which are nothygroscopic and satisfy to the demanding criteria set forth above.

The invention thus concerns a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineand pharmaceutically acceptable solvates thereof.

Sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineand solvates thereof are obtained as powders which have a crystallinephase with good crystallinity and defined morphology. The sulphate saltsof the invention are moreover especially suitable for the preparation ofpharmaceutical compositions containing them. They are pharmaceuticallyacceptable and up to the Applicant's knowledge they are not associatedwith any intreseque toxicity of any kind. In this respect, Applicant hascarried out several in vivo chronic studies with sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineand has not observed any unexpected toxicity event to date.

Moreover, when compared to other salts, such as oxalate salts, the lowermolecular weight of sulphate ions allows for an increased weight ratioofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-aminerelative to the total weight of the salt thereof. For instance, thepercentage by weight of PAM with disulphate salts is of 69%. Therefore,when compared to the oxalate salt, the amount of sulphate salt requiredfor a given dose ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineis reduced. This in return decreases the amount of product administeredto a patient and consequently reduces the production costs ofpharmaceutical compositions containingN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention are sulphate salts of ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineand pharmaceutically acceptable solvates thereof. More particularly, thesulphate salts of the invention and solvates thereof are those ofFormula II

wherein x is 0.5 to 4, preferably x is 0.5 to 3.5, more preferably x is0.9 to 3.In other words, the sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-aminecontains 0.5 to 4 equivalents, preferably 0.5 to 3.5 equivalents, morepreferably 0.9 to 3 equivalents of sulphate for one molecule ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

In one embodiment, x is 2.5 to 3.5, preferably 2.6 to 3.2, morepreferably x is 2.8 to 3, even more preferably x is about 2.9 or x is2.9.

In another embodiment, x is 1.5 to 2.5, preferably x is 1.5 to 2.1, morepreferably x is 1.7 to 1.9, even more preferably x is about 1.8 or x is1.8.

In yet another embodiment, x is 0.5 to 1.5, preferably x is 0.7 to 1.3,more preferably x is 0.9 to 1.1, even more preferably x is about 1 or xis 1.

In another embodiment, x is 1.7 to 2.3, preferably x is 1.9 to 2.1, morepreferably x is about 2 or x is 2.

In a particular embodiment, the sulphate salt of Formula II is in theform of a pharmaceutically acceptable solvate, preferably a hydrate. Thesolvate stoichiometry is between 0.5 to 5, preferably between 1 to 4,more preferably between 1.5 to 2.5, still more preferably y is 1.8 to2.2, even more preferably 2 or about 2 molecules of solvate for 1molecule of sulphate salt of Formula II.

Preferred pharmaceutically acceptable solvates of sulphate salts ofFormula II are those of Formula III

whereinx is as defined above in Formula II, andy is 0.5 to 5, preferably y is 1 to 4, more preferably y is 1.5 to 2.5,still more preferably y is 1.8 to 2.2, even more preferably y is about 2or y is 2.

Preferred compounds of Formula III are those wherein, x is 0.5 to 1.5,preferably 0.8 to 1.2, more preferably x is 0.9 to 1.1, even morepreferably x is about 1 or x is 1.

Particularly preferred compounds of the invention are compounds ofFormula III wherein x is about 1 and y is about 2, or x is 1 and y is 2.

Applicant has shown that a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineand pharmaceutically acceptable solvates thereof are useful forrectifying the metabolism of the Amyloid Protein Precursor (APP) on fouressential points:

-   -   1) increasing the carboxy-terminal fragments of APP (APP-CTFs)        which all in common possess the last 50 amino-acids of APP, and        especially those having potential physiological activities, such        as the α-stubs (APP-CTF alpha) and the γ-stubs (APP-CTF gamma or        AICD for APP intra cellular domain),    -   2) increasing the secretion of sAPP α that presents        neuroprotective/neurotrophic properties,    -   3) decreasing the production of the neurotoxic by-products of        APP, i.e. β-amyloid (Aβ) peptides, especially in their form        x-42,    -   4) without modifying the APP expression and in absence of        neurotoxicity.

The sulphate salts of the invention and solvates thereof are indeeduseful in orienting APP metabolism towards non-amyloidogenic pathways inthe frontal cortex and the hippocampus.

Applicant has also shown that a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineand pharmaceutically acceptable solvates thereof are useful for alteringthe pathological Tau protein phosphorylation while alleviating oxidativestress processes. Tau proteins interact with tubulin to stabilizemicrotubules and promote tubulin assembly into microtubules, microtubulestability being controlled by isoforms and phosphorylation. Taupathologies comprise mechanisms leading to abnormal modifications ofmicrotubule-associated Tau proteins, progressive aggregation andaccumulation into fibrillar material inside degenerating neurons to formthe so-called neurofibrillary tangles (NFT).

In addition, and contrary to other salts, their physico-chemicalproperties are especially useful with regards to drug formulation orsafety, solubility, stability, crystallinity, morphology and toxicity.

The sulphate salts of the invention are thus useful as a medicament, inparticular for treating or preventing neurodegenerative diseases and alldiseases wherein a dysfunction of the APP metabolism is observed,including but not limited to Alzheimer's disease, amyloid angiopathies,dementia with Lewy bodies (DLB) and Down syndrome.

The sulphate salts of the invention are thus useful as a medicament, inparticular for treating or preventing neurodegenerative diseases and alldiseases wherein a dysfunction of the Tau proteins phosphorylation isobserved, including but not limited to tauopathies such asfrontotemporal dementia with Parkinsonism linked to chromosome 17.

Hence, the invention also concerns a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof as defined herein foruse in treating and/or preventing a disease selected fromneurodegenerative diseases including Alzheimer's disease, dementia withLewy bodies (DLB), amyotrophic lateral sclerosis (ALS) withfrontotemporal dementia, inclusion body myopathy with Paget's disease ofbone and/or frontotemporal dementia (IBMPFD), frontotemporal lobardegeneration, synucleopathies, Huntington's disease and Parkinson'sdisease, amyloidopathies including amyloid angiopathies, tauopathiesincluding frontotemporal dementia with Parkinsonism linked to chromosome17, neuromuscular diseases with protein inclusions, as well asdevelopmental diseases including Down syndrome. Preferably, the diseaseis selected from Alzheimer's disease, amyotrophic lateral sclerosis(ALS) with frontotemporal dementia, inclusion body myopathy with Paget'sdisease of bone and/or frontotemporal dementia (IBMPFD), frontotemporallobar degeneration, synucleopathies, Huntington's disease,amyloidopathies including amyloid angiopathies, tauopathies includingfrontotemporal dementia with Parkinsonism linked to chromosome 17. Morepreferably, the disease is selected from Alzheimer's disease,synucleopathies, amyloidopathies including amyloid angiopathies, andtauopathies including frontotemporal dementia with Parkinsonism linkedto chromosome 17. Even more preferably, the disease is selected fromAlzheimer's disease and tauopathies including frontotemporal dementiawith Parkinsonism linked to chromosome 17.

In other terms, the invention also provides for a method of treatingand/or preventing a disease selected from neurodegenerative diseases,amyloidopathies, tauopathies and developmental diseases, in particularthose cited above as well as embodiments thereof, comprisingadministering to a patient in need thereof a pharmaceutically effectiveamount of a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof as described herein. Ina particular embodiment, the disease is selected from Alzheimer'sdisease and tauopathies.

In one particular embodiment, the invention also concerns a sulphatesalt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof as defined herein foruse in delaying in a patient the onset of a disease selected fromneurodegenerative diseases including Alzheimer's disease, dementia withLewy bodies (DLB), amyotrophic lateral sclerosis (ALS) withfrontotemporal dementia, inclusion body myopathy with Paget's disease ofbone and/or frontotemporal dementia (IBMPFD), frontotemporal lobardegeneration, synucleopathies, Huntington's disease and Parkinson'sdisease, amyloidopathies including amyloid angiopathies, tauopathiesincluding frontotemporal dementia with Parkinsonism linked to chromosome17, neuromuscular diseases with protein inclusions, as well asdevelopmental diseases including Down syndrome. Preferably, the diseaseis selected from Alzheimer's disease, amyotrophic lateral sclerosis(ALS) with frontotemporal dementia, inclusion body myopathy with Paget'sdisease of bone and/or frontotemporal dementia (IBMPFD), frontotemporallobar degeneration, synucleopathies, Huntington's disease,amyloidopathies including amyloid angiopathies, tauopathies includingfrontotemporal dementia with Parkinsonism linked to chromosome 17. Morepreferably, the disease is selected from Alzheimer's disease,synucleopathies, amyloidopathies including amyloid angiopathies, andtauopathies including frontotemporal dementia with Parkinsonism linkedto chromosome 17. Even more preferably, the diseases are selected fromAlzheimer's disease and tauopathies including frontotemporal dementiawith Parkinsonism linked to chromosome 17.

In other terms, the invention provides for a method for delaying in apatient the onset of a disease selected from neurodegenerative diseases,amyloidopathies, tauopathies and developmental diseases, in particularthose cited above as well as embodiments thereof, comprisingadministering to a patient in need thereof a pharmaceutically effectiveamount of a sulphate salt of the invention or a pharmaceuticallyacceptable solvate thereof. In a particular embodiment, the disease isselected from Alzheimer's disease and tauopathies.

According to a further feature of the present invention there isprovided a method for modulating APP metabolism, in a patient,preferably a warm blooded animal, and even more preferably a human, inneed of such treatment, which comprises administering to said patient aneffective amount of a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineof the present invention, or a pharmaceutically acceptable solvatethereof.

According to still a further feature of the present invention there isprovided a method for altering pathological Tau protein phosphorylationwhile alleviating oxidative stress processes in a patient, preferably awarm blooded animal, and even more preferably a human, in need of suchtreatment, which comprises administering to said patient an effectiveamount of sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineof the present invention or a pharmaceutically acceptable solvatethereof.

The invention also provides pharmaceutical compositions comprising asulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof as described herein andat least one pharmaceutically acceptable carrier, diluent, excipientand/or adjuvant. In one embodiment, the invention also coverspharmaceutical compositions which contain, in addition to a sulphatesalt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof as active ingredient,additional therapeutic agents and/or active ingredients.

According to one embodiment, the sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineof the invention, as well as their pharmaceutical acceptable solvatesmay be administered as part of a combination therapy. Thus, are includedwithin the scope of the present invention embodiments comprisingco-administration of compositions and medicaments which contain, inaddition to a sulphate salt of the present invention or apharmaceutically acceptable solvate thereof as active ingredient,additional therapeutic agents and/or active ingredients. Such multipledrug regimens, often referred to as “combination therapy”, may be usedin the treatment and/or prevention of any of the diseases or conditionsmediated by or associated with APP metabolism modulation. The use ofsuch combinations of therapeutic agents is especially pertinent withrespect to the treatment of the above-mentioned neurodegenerativediseases within a patient in need of treatment or one at risk ofbecoming such a patient.

In addition to the requirement of therapeutic efficacy, which maynecessitate the use of active agents in addition to the sulphate saltsofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor pharmaceutically acceptable solvates thereof, there may be additionalrationales which compel or highly recommend the use of combinations ofdrugs involving active ingredients which represent adjunct therapy,i.e., which complement and supplement the function performed by thesulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineof the present invention or pharmaceutically acceptable solvatesthereof. Suitable supplementary therapeutic agents used for the purposeof auxiliary treatment include drugs which, instead of directly treatingand/or preventing a disease or condition mediated by or associated withAPP metabolism, treat diseases or conditions which directly result fromor indirectly accompany the basic or underlying APP metabolism modulateddisease or condition.

According to a further feature of the present invention, a sulphate saltofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,a pharmaceutically acceptable solvate thereof may be used in combinationtherapy with other drugs used for treating Alzheimer's disease. Moreparticularly, the compound of Formula II, as well as pharmaceuticallyacceptable solvate thereof, may be used as an adjunct therapy incombination with acetylcholinesterase inhibitors, including but notlimited to donepezil (CAS no 120014-06-4) and salts and solvatesthereof, galantamine (CAS no 357-70-0) and salts and solvates thereof,rivastigmine (CAS no 123441-03-2) and salts and solvates thereof,tacrine (CAS no 321-64-2) and salts and solvates thereof, or incombination with NMDA glutamate receptor antagonists, including but notlimited to memantine (CAS no 19982-08-2) and salts and solvates thereof,or in combination with dual acetylcholinesterase inhibitor and NMDAglutamate receptor antagonist, including but not limited to huperzine A(CAS no 102518-79-6) and salts and solvates thereof, or in combinationwith glucagon-like peptide 1 (GLP-1) agonists, including but not limitedto liraglutide (CAS no 204656-20-2) and salts and solvates thereof,exenatide (CAS no 141732-76-5) and salts and solvates thereof, or incombination with retinoids, including but not limited to acitretin (CASno 55079-83-9) and salts and solvates thereof, or in combination withcalcium channel blockers (CCB), including but not limited to nilvadipine(CAS no 75530-68-6) and salts and solvates thereof, nitrendipine (CAS no39562-70-4) and salts and solvates thereof, nimodipine (CAS no66085-59-4) and salts and solvates thereof or in combination withangiotensin receptor blockers, including but not limited to valsartan(CAS no 137862-53-4) and salts and solvates thereof, or in combinationwith tetracycline antibiotics, including but not limited to minocycline(CAS no 10118-90-8) and salts and solvates thereof.

Thus, the methods of treatment and pharmaceutical compositions of thepresent invention may employ a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof in monotherapy.However, said methods and compositions may also be used multiple therapyin which one or more sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor their pharmaceutically acceptable solvates are co-administered incombination with one or more other therapeutic agents.

In the above-described embodiment, combinations of sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof and other therapeuticactive agents may be administered, in terms of dosage forms, eitherseparately or in conjunction with each other, and in terms of their timeof administration, either serially or simultaneously. Thus, theadministration of one component agent may be prior to, concurrent with,or subsequent to the administration of the other component agent(s).

Generally, for pharmaceutical use, the sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor pharmaceutically acceptable solvates thereof may be formulated as apharmaceutical composition comprising at least one sulphate salt of theinvention or a pharmaceutically acceptable solvate thereof and at leastone pharmaceutically acceptable carrier, diluent, excipient and/oradjuvant, and optionally one or more further therapeutic agents and/oractive ingredients.

By means of non-limiting examples, pharmaceutical composition may be ina dosage form suitable for oral administration, for parenteraladministration (such as by intravenous, intramuscular or subcutaneousinjection or intravenous infusion), for topical administration(including ocular), for administration by inhalation, by a skin patch,by an implant, by a suppository, etc. Such suitable administrationforms—which may be solid, semi-solid or liquid, depending on the mannerof administration—as well as methods and carriers, diluents andexcipients for use in the preparation thereof, will be clear to theskilled person; reference is made to the latest edition of Remington'sPharmaceutical Sciences. The pharmaceutical compositions may beformulated in solid form and re-dissolved or suspended prior to use.

Some preferred, but non-limiting examples of dosage forms includetablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols, ointments, cremes,lotions, soft and hard gelatin capsules, suppositories, drops, sterileinjectable solutions and sterile packaged powders (which are usuallyreconstituted prior to use) for administration as a bolus and/or forcontinuous administration, which may be formulated with carriers,excipients, and diluents that are suitable per se for such formulations,such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, alginates, tragacanth, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethyleneglycol, cellulose, (sterile) water, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetableoils and mineral oils or suitable mixtures thereof. The pharmaceuticalcompositions can optionally contain other substances that are commonlyused in pharmaceutical formulations, such as lubricating agents, wettingagents, emulsifying and suspending agents, dispersing agents,disintegrating agents, stabilizing agents, isotonic agents, bulkingagents, fillers, preserving agents, sweetening agents, flavouringagents, perfuming agents, colouring agents, antibacterial agents and/orantifungal agents such as parabens, chlorobutanol, phenol, sorbic acid,dispensing agents, flow regulators, release agents, etc. Thecompositions may also be formulated so as to provide rapid, sustained ordelayed release of the active compound(s) contained therein.

The pharmaceutical compositions of the invention are preferably in aunit dosage form, and may be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which may be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 0.05 and 1000 mg, and usually between 1 and 500 mg,of the at least one compound of the invention, e.g. about 10, 25, 50,100, 200, 300 or 400 mg per unit dosage.

Usually, depending on the condition to be prevented or treated and theroute of administration, the active compound of the invention willusually be administered between 0.01 to 100 mg per kilogram, more oftenbetween 0.1 and 50 mg, such as between 1 and 25 mg, for example about0.5, 1, 5, 10, 15, 20 or 25 mg, per kilogram body weight of the patientper day, which may be administered as a single daily dose, divided overone or more daily doses, or essentially continuously, e.g. using a dripinfusion.

All references to compounds of Formula II include references tosolvates, in particular compounds of Formula III, multi-componentcomplexes and liquid crystals thereof.

The compounds disclosed throughout the present application were namedusing ChemDraw® Ultra version 12.0 (CambridgeSoft, Cambridge, Mass.,USA).

N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-aminecan be obtained as disclosed in WO2006/051489. The sulphate salts andsolvates thereof can be prepared according to techniques known in theart such as those involving precipitation, crystallization,recrystallization, lyophilisation, phase transfer or ion exchangeresins.

Definitions

The term “solvate” is used herein to describe a compound in thisinvention that contains stoichiometric or sub-stoichiometric amounts ofone or more pharmaceutically acceptable solvent molecule such asethanol. The term “hydrate” refers to when the said solvent is water.The pharmaceutically acceptable solvent molecules may be co-crystallizedwith the compound of the invention, and/or be present in crystallineand/or amorphous phases of solids thereof, and/or be adsorbed thereto.

The term “Alzheimer's disease” as used herein, designates all types ofAlzheimer's disease, including but not limited to the sporadic andfamilial types.

The term “inclusion body myopathy with Paget's disease of bone and/orfrontotemporal dementia (IBMPFD)” as used herein, is a type of myopathy,more specifically an inherited adult onset multisystem disease thataffects muscle, bone and the central nervous system. Patients with thiscondition can present with a variety of manifestations, comprisinginclusion body myopathy, Paget's Disease of the bone, frontotemporaldementia and/or amyotrophic lateral sclerosis (Lou Gehrig's disease).

The term “dementia with Lewy bodies (DLB)” as used herein, also known asLewy body dementia, diffuse Lewy body disease, cortical Lewy bodydisease and senile dementia of Lewy type, is a type of dementia closelyassociated with both Alzheimer's and Parkinson's diseases. It ischaracterized by the presence of Lewy bodies, clumps of alpha-synucleinand ubiquitin protein in neurons, detectable in post mortem brainhistology.

The term “synucleopathies” as used herein means a disease of the centralnervous system characterized by alpha synuclein-positive depositions inneurons.

The term “amyloid angiopathies” as used herein means diseases related toamyloid deposits forming in the walls of the blood vessels of thecentral nervous system.

The term “tauopathies” as used herein means neurodegenerative diseasesassociated with the pathological aggregation of tau protein in the humanbrain.

The term “developmental diseases” as used herein means any conditionthat appears at some stage in a child's development and delays orprevent the development of one or more physiological functions such aslanguage skills Developmental diseases include psychological andphysical diseases. Non-limiting examples of developmental diseases areautism spectrum disorder (ASD), Down syndrome, attention deficitdisorder (ADD) and attention deficit hyperactive disorder (ADHD).

The term “patient” refers to a warm-blooded animal, more preferably ahuman, who/which is awaiting the receipt of, or is receiving medicalcare or is/will be the object of a medical procedure.

The term “human” refers to a subject of both genders and at any stage ofdevelopment (i.e. neonate, infant, juvenile, adolescent, adult).

The terms “treat”, “treating” and “treatment, as used herein, are meantto include alleviating, attenuating or abrogating a condition or diseaseand/or its attendant symptoms.

The terms “prevent”, “preventing” and “prevention”, as used herein,refer to a method of delaying or precluding the onset of a condition ordisease and/or its attendant symptoms, barring a patient from acquiringa condition or disease, or reducing a patient's risk of acquiring acondition or disease.

The term “therapeutically effective amount” (or more simply an“effective amount”) as used herein means the amount of active agent oractive ingredient (e.g.N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine)that is sufficient to achieve the desired therapeutic or prophylacticeffect in the patient to which/whom it is administered.

The term “administration”, or a variant thereof (e.g., “administering”),means providing the active agent or active ingredient (e.g.N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine),alone or as part of a pharmaceutically acceptable composition, to thepatient in whom/which the condition, symptom, or disease is to betreated or prevented.

By “pharmaceutically acceptable” is meant that the ingredients of apharmaceutical composition are compatible with each other and notdeleterious to the patient thereof.

The term “pharmaceutical vehicle” as used herein means a carrier orinert medium used as solvent or diluent in which the pharmaceuticallyactive agent is formulated and/or administered. Non-limiting examples ofpharmaceutical vehicles include creams, gels, lotions, solutions, andliposomes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DSC spectrum of the tartrate salt (1 eq) ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

FIG. 2 shows the DSC spectrum of the fumarate salt (1 eq) ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

FIG. 3 shows the DSC spectrum of the oxalate salt (2 eq) ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

FIG. 4 shows the DSC spectrum of the oxalate salt (3 eq) ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

FIG. 5 shows the DSC spectrum of the oxalate salt (4 eq) ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

FIG. 6 shows the DSC spectrum of the sulphate salt (1 eq) ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

FIG. 7 shows the DSC spectrum of the sulphate salt (2 eq) ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine.

FIG. 8 shows the XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,4 eq oxalate salt.

FIG. 9 shows the XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,2.9 eq sulphate salt of example 1.

FIG. 10 shows the XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1.8 eq sulphate salt of example 2 (higher intensity graph) superimposedwith the one ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,2.9 eq sulphate salt of example 1 (lower intensity graph).

FIG. 11 shows the XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq sulphate salt of example 3.

FIG. 12 shows the XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq sulphate salt of example 3 (bottom graph) superimposed with the oneofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(free base) (top graph).

FIG. 13-a shows photographs of crystals ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine1 eq sulphate salt, as observable with an optical microscope, at zoomx325, under transmitted light.

FIG. 13-b shows photographs of crystals ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine1 eq sulphate salt, as observable with an optical microscope, at zoomx325, under cross polarized light.

FIG. 14 shows the XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,2 eq sulphate salt of example 4.

FIG. 15 shows the XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,2 eq sulphate salt of example 4 (bottom graph) superimposed with the oneofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(free base) (top graph).

FIG. 16 shows the DVS drying curve at 25° C. ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq sulphate salt of example 3.

FIG. 17 shows the DVS isotherm sorption/desorption plots at 25° C. ofN-(3-(4-(3-(diisobutylamino)propyl)perazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq sulphate salt of example 3.

FIG. 18 shows the DVS isotherm sorption/desorption plots at 25° C. ofthe di-hydrated form ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq sulphate salt of example 3.

FIGS. 19-a to 19-d show the results obtained forN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1.8 eq sulphate salt in the in vitro APP metabolism assay. In allfigures, the results are provided for the control (white bar) and thesulphate salt of the invention at four concentrations (black bars). FIG.19-a shows the AICD levels, FIG. 19-b the CTFαlevels, FIG. 19-c theAβ₁₋₄₂ levels and FIG. 19-d the sAPPα levels.

FIG. 20 shows the CTFα levels measured in the frontal cortex of micetreated with various doses ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1.8 eq sulphate salt (in vivo APP metabolism assay; 24 h acutetreatment). The results are provided for the vehicle (white bar) and thesulphate salt of the invention at five doses (dark grey bars).

FIG. 21 shows the CTFα levels measured in the frontal cortex of micetreated with various doses ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,2 eq sulphate salt or of the oxalate salt of WO2006/051489 (in vivo APPmetabolism assay; mice 24 h acute treatment). The results are providedfor the vehicle (white bar), the oxalate salt (light grey bars) and thesulphate salt of the invention (dark grey bars) at 6 mg/kg.

FIGS. 22-a and 22-b show the results obtained forN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1.8 eq sulphate salt in the in vivo APP metabolism assay (rats 1-monthchronic study). In all figures, the results are provided for the vehicle(white bar) and the sulphate salt of the invention at two concentrations(dark grey bars). FIG. 22-a shows the CTF levels in the frontal cortexand FIG. 22-b the CTFβ levels in the hippocampus.

FIGS. 23-a, 23-b and 23-c show the results obtained forN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1.8 eq sulphate salt in the in vivo APP metabolism assay (mice 3-monthschronic study). In all figures, the results are provided for the vehicle(white bar) and the sulphate salt of the invention at threeconcentrations (dark grey bars). FIG. 23-a shows the AT100 levels, FIG.23-b the AT8 levels, and FIG. 23-c the LPO levels.

CHEMISTRY EXAMPLES

The following abbreviations are used throughout the present application:° C.: Celsius degrees, DIPE: di-iso-propyl ether, DSC: differentialscanning calorimetry, DVS: dynamic water vapour sorption/desorption, δ:NMR chemical shifts expressed in ppm, eq: equivalent(s), Et: ethyl, g:gram(s), h: hour(s), HPLC: high performance liquid chromatography, IPA:isopropanol, IR: infrared, L: liter(s), LCMS: HPLC coupled to a massspectrometer, M: mol/L, mM: mmol/L, μM: μmon, Me: methyl, mg:milligram(s), min: minute(s), mL: milliliter(s), mol: mole(s), mmol:millimole(s), μmol: micromole(s), MS: Mass Spectrometry, NMR: nuclearmagnetic resonance, ppm: party per million, RH: relative humidity, rm:reaction mixture, rpm: round(s) per minute, rt: retention time, RT: roomtemperature (ca 15-25° C.), RV: reaction vessel, THF: tetrahydrofuran,XRPD: X ray powder diffraction, W: Watt(s).

All reported temperatures are expressed in degrees Celsius (° C.); allreactions were carried out at room temperature (RT) unless otherwisestated.

Experimental set-up or purification procedures that were used in thisinvention, when not described in specific details, are assumed to beknown to those conversant in the art and are described in such standardreference manuals as: i) Gordon, A. J.; Ford, R. A. “The Chemist'sCompanion—A Handbook of Practical Data, Techniques, and References”,Wiley: New York, 1972; ii) Vogel's Textbook of Practical OrganicChemistry, Pearson Prentice Hall: London, 1989.

HPLC analysis.

Method A:

HPLC spectra were typically obtained on a Waters Alliance 2695 systemHPLC. The instrument includes an autosampler, a quaternary pump, and anultraviolet multi-wavelength detector. The chromatography column usedwas a Waters X-Terra RP18 5 μm, 4.6×250 mm.

Eluent typically used was a mixture of solution A (0.1% TFA in H₂O) andsolution B (0.1% TFA in MeOH).

Gradient was applied at a flow rate of 1 mL per minute as follows:gradient held the initial conditions of 5% solution B for 0 min,increased linearly to 40% solution B in 50 min, held at 40% during 5min, returned to initial conditions in 1 min and maintained for 5 min.

Method B:

In a variant, HPLC analyses were carried out according to the parametersdisclosed in Table 1 below.

TABLE 1 HPLC parameters for method B HPLC equipment Injector/pump:Waters Alliance 2695 Detector: Waters Photo Diode Array 996 Software:Waters Millenium Column SymmetryShield C18 150 mm × 4.6 mmm − dp = 5 μmMobile phase A: H₂O/TFA 0.05% B: MeOH/TFA 0.05% Time A % B % 0 95 5 5 955 20 10 90 25 10 90 25.1 95 5 30 95 5 Flow rate 1 mL/min Columntemperature Room temperature Detection UV: λ = 276 nm Test solutionSuitable dilution in H₂O/CH₃CN 50/50 (v/v) Injection volume 5 μLInjector temperature 20° C. Retention time Free base ~13 min

NMR Analysis

¹H (300 MHz) spectra were recorded on a Bruker Advance DRX 300 MHzinstrument.

Chemical shifts are expressed in parts per million, (ppm, δ units).Coupling constants are expressed in Hertz (Hz). Abbreviations formultiplicities observed in NMR spectra are as follows: s (singlet), d(doublet), t (triplet), q (quadruplet), m (multiplet), br (broad).

DSC spectra. TA Instrument, DSC Q10.

DSC spectra were recorded on a TA DSC Q10 instrument within atemperature range of −10° C. to 300° C. or 400° C. and with 10° C.increments.

Optical Microscopy.

Observation by optical microscopy was performed on a LEICA DMIRBmicroscope equipped with a digital camera and a motorized stage. A fewpowder grains are dispersed on a glass plate with mineral oil. Photoswere recorded with a picture analysis platform from MicrovisionInstruments, both under transmitted light and polarized light.

X-Ray Powder Diffraction

X-ray powder diffraction (XRPD) analysis is performed on a Brüker—AXS D8Advance diffractometer, using a copper anti-cathode, a mono-crystallinesilicon sample holder and a position sensitive detector. Instrumentoperating conditions for X-rays pattern acquisition are described inTable 2.

TABLE 2 Instrument operating conditions for X-rays profile acquisitionTemperature Ambient Atmosphere Ambient X-rays generator voltage (kV) 40intensity (mA) 40 X-rays source target Cu emission radiation Kλ₁ (nm)0.15406 Kλ₂ (nm) 0.15444 Ratio Kλ₂/Kλ₁ 0.5 Kβ filter Ni Slit (nm)anti-divergence 0.6 Goniometer angular sector analyzed (° for 2θ) 5-70step size (° for 2θ) 0.0714 Rotation speed for sample holder (rpm) 30Detection angular opening (θ) 8 step time for measuring diffracted 6intensity (s)

The powder sample is dispersed on the silicon sample holder in a way toavoid preferred orientation (not randomly oriented crystals) and toensure planarity of the specimen surface.

Dynamic Vapour Sorption

Dynamic vapour sorption (DVS) analyses with water were performed on aDVS-Intrisic incubator from SMS Ltd, equipped with DVS-Intrisic ControlSoftware 1.0.

A sample of about 5 to 10 mg, placed in an aluminium pan holder, wassubmitted to a full-cycle analysis (sorption followed by desorption)under the conditions described in Table 3.

TABLE 3 operating conditions for DVS analysis Temperature 25° C. Carriergas and rate Dried and filtered air at 100 mL · min⁻¹ Mode and criteriondm/dt ≤0.002% · min⁻¹ Humidity range 0 to 95% RH RH step 5% Minumum steptime 10 min Maximum step time 360 min

The sample was pre-dried under a stream of dry filtered air until astable mass was obtained. Relative humidity was then increased by 5%increments. At each step, the sample was allowed to increase untilequilibrium is reached (dm/dt criterion), then relative humidity wasincreased further. Relative humidity was ramped up to 95%. Afterequilibration at this stage, desorption is started in a similar stepwisemanner, with a sample weight allowed to stabilize after each incrementalhumidity decrease step.

Solvents and reagents were purchased and used as received fromcommercial vendors unless otherwise specified.

Example 1: Synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)perazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,2.9 eq Sulphate Salt

A diluted sulphuric acid solution in DIPE was prepared by adding 4 mL(0.0751 mol) of concentrated sulphuric acid to 28 mL (0.197 mol) ofDIPE.

To a suspension ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(16.1 g, 0.03756 mol) in DIPE (280 mL), was added dropwise 32 mL of theabove-described diluted sulphuric acid solution. A slight temperatureincrease around 10° C. was observed. The reaction mixture was stirred atRT and became limpid over an hour. The resulting white solution wasfiltered and recovered. The white powder obtained was then dried undervacuum at 50° C. during 48 hours.

Analytical Data

HPLC (method A): rt: 20.24 min;

Elemental analysis:

-   -   calculated: % C=48.06; % H=7.74; N=13.45; % S=10.26; %0=20.48    -   experimental: % C=41.43; % H=6.95; % N=11.60%; % S=12.34;        %0=26.53.

Example 2: Synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1.8 eq Sulphate Salt

Title compound was prepared according to a similar procedure as the onedescribed at example 1.

Analytical Data

HPLC (method A): rt: 29,207 min;

NMR (D₂O): δ(ppm): 7.2 (m, 4H, 2CH); 3.7 (b, 8H, 4CH ₂); 3.4-3.1 (m, 8H,4CH₂); 2.9 (m 4H, 2CH ₂); 2.3-1.9 (m, 6H, 2CH+2CH ₂); 0.85 (d, 12H,4CH₃).

Elemental analysis:

-   -   calculated: % C=48.06; % H=7.74; N=13.45; % S=10.26; %0=20.48    -   experimental: % C=49.55; % H=7.89; N=13.87; % S=8.95; %0=19.29.

Example 3: Synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq Sulphate Salt

3 mL of ethanol were added to 99.6 mg of synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,almost complete dissolution was achieved upon heating at 70° C. Anequimolar quantity of a 0.5 M aqueous solution of sulphuric acid wasadded (465 μL) and full dissolution was observed. The solution is driedin vacuo at 70° C. to give a solid residue. The solid is re-suspended inIPA/EtOH at 70° C. to provide a suspension of fine particles; thesolvent volume was then partially reduced under vacuum. The suspensionwas allowed to cool to room temperature. The supernatant was removed andthe powder was dried under vacuum for 2 h at 70° C., to yield titlecompound in 89% yield.

HPLC analyses (method B): the percentage ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(free base) in title compound, determined by external standardization,was found to be 83.4%. This result was consistent with the theoreticalcalculated percent: 81.4%.

Example 4: Synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,2 eq Sulphate Salt

3 mL of ethanol were added to 101.7 mg of synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,almost complete dissolution was achieved upon heating at 70° C. Atwo-molar quantity of a 0.5 M aqueous solution of sulphuric acid wasadded (950 μL) and full dissolution was observed. The solution is driedin vacuo at 70° C. to give a translucent film wherein crystallization ofexpected salt progressively occurred. The solid was then re-suspended in3 mL of methanol to give a clear suspension which is stirred at 60° C.for 15 minutes. 10 mL of IPA were added to finalize salt crystallizationand the resulting suspension was stirred at 80° C. for 30 minutes. Thesuspension was then allowed to cool to room temperature and storedovernight at sub-ambiant temperature. The supernatant was removed fromthe flask and the powder dried in vacuo for 2.5 hours at 70° C. toprovide title compound in 71% yield.

HPLC analyses (method B): the percentage ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(free base) in title compound, determined by external standardization,was found to be 65.8%. This result was consistent with the theoreticalcalculated percent: 68.6%.

No long term instability due to oxidation, in particular oxidation atthe piperazine ring, was observed with sulphate salts in solid state.

Example 5: Physico-Chemical Analysis of Different Salts

The hydrochloride, hydrobromide, acetate, tartrate, fumarate, malate,oxalate and sulphate salts ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-aminewere prepared according to procedures similar to those described in theprevious examples or standard salt formation methods well known in theart. The results of these salt formations are reported in Table 4.

TABLE 4 salt formation results Salt Acid eq. added Physical form Freebase 0 Amorphous powder Chloride 4 Precipitation of a very hygroscopicsolid ¹ Bromide 1.1 No solid formed Acetate 1 No solid formed, oilyresidue Acetate 2 No solid formed, oily residue Fumarate 1 Notcrystalline² Tartrate³ 1 Not crystalline² Malate 1 Powder Oxalate 2 GumOxalate 3 Gum Oxalate 4 Crystalline powder Sulphate 1 Crystalline powder(colorless solid) Sulphate 2 Crystalline powder (colorless solid) ¹ aweighed out sample of chloride salt was left standing open to the air.Said sample rapidly gained weight and eventually became deliquescentwithin a few seconds or minutes, depending on the room RH. ²DSC spectrumdid not reveal any endotherm and is thus characteristic of solidslacking a crystalline phase ³salt formed in EtOH/water

The chloride salt was straightforwardly found very hygroscopic whileacetate salts were not obtained in solid form. Although tartrate andfumarate salts were obtained as solids, the DSC analysis showed thatthey are not crystalline (see FIGS. 1 and 2). Such deficiency can resultin batch to batch variations with respect to solid forms. Batch to batchreproducibility is an essential criterion in the synthesis of drugcompounds especially when different solid forms can have differentpharmacokinetics and pharmacodynamics properties. Hence, tartrate andfumarate salts are not satisfactory.

The malate salt was obtained as a powder; however it bears an asymmetriccarbon. Chiral centers considerably complicate clinical developmentsince each stereoisomer needs to be equally characterized as the activestereoisomer.

With regards to oxalate salts, 4 eq of oxalate counter-ions are requiredto obtain suitable crystalline powders. The DSC graphs of oxalate salts(2 eq, 3 eq and 4 eq) are shown respectively in FIGS. 3, 4 and 5. Asshown by the DSC spectra the tetraoxalate salt bear the most well shapedand defined endotherm. The requirement of having 4 equivalents ofoxalate salt is a caveat since the weight percentage of active moleculedrops to as low as 54%. The amount of drug to be administered for agiven dose of PAM is thus significantly increased as well as the risk ofnephrotoxicity associated to oxalates.

Both sulphate salts precipitated as solids. They were found stable whenleft open to the air. In contrast to the chloride salt, nohygroscopicity issue was observed. The DSC analysis (see FIGS. 6 and 7)revealed well defined endotherms which are consistent with solidscomprising a crystalline phase.

Example 6: XRPD Analysis of Oxalate and Sulphate Salts

The XRPD diffractogram ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,4 eq oxalate salt is shown on FIG. 8. In the angular window analyzed, afew large diffraction rays of low intensity were detected. The XPRDprofile reveals the presence of a high diffusion background. Such a XPRDprofile is characteristic of a powder having a crystalline phase withlittle or poor crystallinity (large rays) with the presence of anamorphous phase (high diffusion background).

The X-Ray diffraction profile for the sulphate salt of example 1 (seeFIG. 9) shows several well-defined and sharp diffraction peaks between 5and 25° in 2-theta scale, indicating that the solid form of saidsulphate salt comprises a well good crystalline phase. The diffractogramalso presents a background halo (between 10 and 30°) that is interpretedas coming from an amorphous fraction present in the solid. Profilescomparison with the sulphate salt of example 2 reveal many overlappingbetween the several diffraction peaks detected (see FIG. 10).

The XPRD diffractogram of the sulphate salt of example 3 (see FIG. 11)confirmed the good crystallinity of this salt with numerous, fine andwell defined diffraction peaks between 4 and 30° 2θ, and reported adifferent diffraction profile from synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(free base). The XPRD profile of this salt has been compared with theone of the free base, both profiles superimposed in a same chromatograph(FIG. 12) are consistent with different crystal forms between thesulphate salt and the free base. These results are consistent with theobservations by optical microscopy. Indeed, the high birefringence andthe well defined stick-like morphology of particles when observed undertransmitted light (FIG. 13a ) and cross polarized light (FIG. 13b )indicated a sulphate salt of good crystallinity.

The XPRD diffractogram of the sulphate salt of example 4 (see FIG. 14)confirmed the good crystallinity of this salt with numerous diffractionpeaks between 4 and 30° 2θ, and reported a different diffraction profilefrom synthesis ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(free base). The XPRD profile of this salt has been compared with theone of the free base, both profiles superimposed in a same chromatograph(FIG. 15) are consistent with different crystal forms between thesulphate salt and the free base.

As a result, Examples 5 and 6 clearly show that sulphate salts aresuperior to any other acid salt and to oxalate salts in particular.Sulphate salts do not bear severe hygroscopicity as chloride salts.Unlike bromide salts, they crystallize to provide powder materials.Their crystallinity is indeed far superior to those of tartrate andfumarate salts. Like sulphate salts, the oxalate salt with a specific 4eq stoichiometry provides solids that comprise a crystalline and anamorphous phase. However, the XRPD profiles and optical microscopy undertransmitted and polarized lights revealed that the crystalline phase ofsulphate salts is better defined in terms of morphology andcrystallinity than the one of the oxalate salt. Moreover, sulphate saltsremedy the shortcomings ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine(free base), i.e. they can be isolated in a solid form having acrystallized phase with a good crystallinity and have no obviousstability issues. Furthermore, they broaden the formulationpossibilities of this PAM owing to their very high water solubility. The4 eq oxalate salt did not fully remedy the shortcomings of the free basein terms of crystallinity and morphology and are anyway inferior tosulphate salts in these aspects. The skilled person would even considera 4 eq oxalate salt as worsening the overall properties of the free basePAM since it contains high quantities of oxalate counter-ions which caninduce severe nephrotoxicity.

Example 7: DVS Profile ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq Sulphate Salt

DVS analysis was carried out according to the method described above.

The results are shown in FIGS. 16 and 17 which display the DVS dryingcurve as well as the DVS isotherm plots (water vapoursorption/desorption traces) after preliminary drying of the sulphatesalt of example 3. In FIG. 17, the top curve corresponds to thedesorption phase and bottom curve to the sorption phase.

Upon drying at 25° C./0% RH, the sample lost 0.18% of its mass.

Upon sorption, three relative humidity intervals corresponding todifferent water sorption rates and behaviours were observed from thesample mass variation:

-   -   from 0 to 80% RH: continuous and slow water sorption rate; water        uptake at 25° C./60% RH and 25° C./80% RH were respectively of        0.7% and 1.5% compared to the sample mass obtained after initial        drying at 0% RH;    -   from 80 to 90% RH: rapid and nearly constant water sorption        rate; water uptake at 25° C./90% RH was 9.7% compared to the        sample mass obtained after initial drying at 0% RH;    -   water sorption then further increased and water vapour uptake at        25° C./95% RH was of 19.4% compared to the sample mass obtained        after initial drying at 0% RH.

Upon desorption, three relative humidity intervals corresponding todifferent water sorption rates and behaviours were observed:

-   -   from 95 to 90% RH: rapid decrease of the residual water rate        with 10% remaining at 25° C./75% RH compared to the sample mass        obtained after initial drying at 0% RH;    -   from 75 to 10% RH: the sample retained the “adsorbed” water with        a strong and nearly constant hysteresis (up to 6.7% at 25°        C./75% RH);    -   from 10 to 0% RH: the sample lost part of its water; the        remaining amount of water was 4.6% at 25° C./0% RH, compared to        the sample mass obtained after initial drying at 0% RH; at this        stage the sample mass was stabilized and thus in equilibrium        with the surrounding environment.

Based on commonly used criteria (i.e. a compound is said hygroscopic ifit presents more than 2% by weight water uptake at 25° C./60% RH), thesulphate salt of example 3 was not hygroscopic at 25° C./60% RH, withonly an overall mass uptake at this stage.

Given the rapid water uptake at 80% RH, as well as the strong hysteresis(6 to 6.7% of water remaining from 80 to 10% RH upon desorption), thesulphate salt of example 3, originally obtained as its non-solvatedform, converted into a di-hydrated form at high RH. The theoreticalwater uptake for the di-hydrated form ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq sulphate salt of example 3 is 6.8% and in line with experimentallydetermined 6.7%.

At the end of the above-described first DVS cycle, the same sample wassubmitted to a second DVS cycle with a second sorption phase and asecond desorption phase. The results are shown in FIG. 18. Both secondsorption and desorption phases were identical to the first desorptionphase curve obtained during the first DVS cycle. Indeed, as can be seenin FIG. 18, desorption phase and sorption phase overlap each other. Thedi-hydrated form ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine,1 eq sulphate salt of example 3, which was obtained beyond 80% RH at 25°C. during the first cycle, was thus stable and non hygroscopic.

Biology Examples

In Vitro APP Metabolism Assay

This assay was performed using SH-5Y5Y cells (human neuroblastoma cellline overexpressing wild type human APP) which were treated for 24 hwith the sulphate salt of example 2. This test was carried out at fourdifferent compound concentrations of 0.3, 1, 3 and 10 μM. Quantificationof different metabolites was achieved by western-blot analysis. CTFα,CTFβ and AICD were analysed using anti-actin ((I-19) (SC-1616 Santa CruzBiotechnology; 1:1000 from 200 μg/ml stock) as an internal control andanti-C term APP antibody (1:250000 in washing buffer). Proteinconcentrations were determined by the BCA Protein Assay Kit (ThermoScientific). Samples (20 μg total protein) were separated by 16%SDS-PAGE according to the molecular weight of each protein, andtransferred to nitrocellulose membranes.

The results are shown on FIGS. 19-a, 19-b, 19-c and 19-d. They show dosedependent increased levels of AICD, CTFα and sAPPα and decreased levelof Aβ₁₋₄₂.

Sulphate salt of example 2 thus induces a significant increase of APPmetabolism through non-amyloidogenic pathway concomitant with areduction in the formation of deleterious metabolites involved inamyloid plaques formation. These results are all the more so remarkablesince Aβ₁₋₄₂ is one of the most deleterious APP metabolism by-productswith respect to amyloid plaques formation. Furthermore, peptide sAPPαdoes not induce amyloid plaque formation and is rather recognized ashaving beneficial neuroprotective effects.

In addition, it is remarked that no cytotoxicity was found for thissulphate salt, its CC₅₀ on SH-5Y5Y cell line was found greater than 30μM. CC₅₀ is defined herein as the concentration at which 50% of platedcells remain alive.

In Vivo APP Metabolism Assay

In vivo experiments were carried out on 4-month old C57B16 female miceor 2-month old Sprague Dawley rats.

Acute treatment: 24 h p.o.

C57B16 female mice were treated per os (gavage) with vehicle or compound(example 2) at 0.25, 0.5, 1, 3 and 6 mg/kg mg/kg (n=6 per group) for 24h. The product was administered with a disposable Rodent Feeding TubeECIMED Ref# V0104030 (4 mm×30 mm). The animals were sacrificed after 24h, the brain was immediately removed for dissection. Levels of CTFα inthe frontal cortex (FC) and/or the hippocampus (HIP) were measured bywestern-blot analysis as previously described. Briefly, tissues werehomogenized with 200 μL of lysis buffer (10 mL of Laemmli Pre-LysisBuffer and 1 tablet of Protease Inhibitor cocktail Complete Mini(Roche)) in a potter. After sonication (5 min), homogenates werecentrifuged at 1600 rpm 5 min 4° C. Supernatants were aliquoted andstored at −80° C. before western blot analysis.

Dose dependent results are shown on FIG. 20. Compound of example 2enhances APP metabolism through the non-amyloidogenic pathway in thefrontal cortex as shown by increasing concentrations of CTFα. Thefrontal cortex is generally the first region of the brain to be affectedby amyloid plaques formation in abnormal APP metabolism relateddiseases.

The compound of example 2 was also compared to the oxalate salt ofWO2006/051489 in the same assay (6 mg/kg dose). The results shown inFIG. 21 indicate superior efficiency of the sulphate salt in comparisonto the oxalate salt.

Chronic Treatment: 1-Month

Rats were provided for one month with 1 or 10 mg/kg/day of the compoundof example 2 dissolved in their drinking water. The remarkable highsolubility of this sulphate salt enabled easy formulation andadministration thereof to the animals. The animals were sacrificed afterone month and levels of CTFα and CTFβ in the frontal cortex (FC) and/orthe hippocampus (HIP) were measured by western-blot analysis aspreviously described. Dose dependent results are shown on FIGS. 22-a and22-b. Sulphate salt of example 2 enhances APP metabolism through thenon-amyloidogenic pathway in the frontal cortex as shown by increasingconcentrations of CTFα (FIG. 22-a). Sulphate salt of example 2attenuates APP metabolism through the amyloidogenic pathway in thehippocampus as shown by decreasing concentrations of CTFβ (FIG. 22-b).The frontal cortex is generally the first region of the brain to beaffected by amyloid plaques formation in abnormal APP metabolism relateddiseases while the hippocampus, which is highly involved in memory andrecollection processes, is latterly but severely affected. The sulphatesalt of example 2 thus has very positive effect on APP metabolism inboth the frontal cortex and the hippocampus and could is therefore ofinterest in the treatment of neurodegenerative diseases, in particularfor both early and advanced stages of abnormal APP metabolism relateddiseases.

Chronic Treatment: 3-Months

Neuropathological disorders are also characterized by abnormalphosphorylation of Tau protein (AT100). Hyperphosphorylation of the tauprotein (on specific sites) can result in the intracellular accumulationof neurofibrillary tangles (NFTs), involved in the pathogenesis ofAlzheimer's disease and other tauopathies. One axis of the study wasthus to study the effect of the sulphate salt of example 2 on thisabnormal phosphorylation. In parallel, such effect was also monitoredwith regards to the non-pathological phosphorylation of Tau protein(AT8) which should remain unaffected. Another axis of this study wasthus the impact of the sulphate salt of example 2 on oxidative stress(OS). Therefore, levels of lipidic peroxidation, a well known marker forthe evaluation of oxidative stress, were determined. Indeed, oxidativestress (OS), by the generation of toxic reactive oxygen species (ROS)and oxidative damage (oxidation of vital cellular components as lipids,proteins and DNA), is believed to be involved in the pathogenesis ofneurodegenerative disorders. The neuronal cell OS response isparticularly studied for its contribution to the neurodegenerationprocesses. OS results from a misbalance between ROS generation andantioxidant defences, leading to an accumulation of oxidative damages,and finally the cell death. Oxidative damage has also been associatedwith pathological neuronal loss in Parkinson's disease (PD) andHuntington's disease (HD).

4-month old C57B16 female mice were provided with 0.5, 1 or 3 mg/kg ofcompound of example 2 dissolved in their drinking water. First of all,all mice were weighed and distributed in each cage in order to haveapproximately the same mean of weight ±SD per cage. Each productconcentration was prepared in sterile bottles and kept at RT protectedfrom light. Drinking bottles were filled each week and weighed. Volumeconsumed was calculated by weighing each bottle after each week and theremaining volume was discarded.

AT100 phosphorylation levels measurements were performed on braintissues by western blot analysis (previously described) using specificanti AT100 antibody (Anti-human PHF-Tau monoclonal antibody, MN1020,ThermoScientific/Pierce).

AT8 phosphorylation levels measurements were performed on brain tissuesby western blot analysis (previously described) using specific anti AT8antibody (Anti-human PHF-Tau monoclonal antibody, MN1060,ThermoScientific/Pierce).

LPO levels measurements: the level of lipid peroxidation in hippocampiis determined as cumene hydroperoxide (CHP) equivalents and expressed asCHP equivalents per wet weight of tissue and as percentage of controlgroup data following the modified FOX assay.

The results are shown in FIGS. 23-a, 23-b and 23-c. The sulphate salt ofthe invention decreases the pathological Tau protein phosphorylation(FIG. 23-a) while not affecting the normal Tau protein phosphorylation(FIG. 23-b). Furthermore, this sulphate salt induces a significantdecrease of LPO levels and is thus able to partly alleviate oxidativestress processes (FIG. 23-c).

In view of the above experimental results, sulphate salts of theinvention are useful in orienting APP metabolism towardsnon-amyloidogenic pathways in the frontal cortex and the hippocampus.They further alter the pathological Tau protein phosphorylation whilealleviating oxidative stress processes.

The invention claimed is:
 1. A method for delaying the onset of ortreating a disease selected from Alzheimer's disease and Parkinson'sdisease comprising administering to a patient in need thereof apharmaceutically effective amount of a sulphate salt ofN-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amineor a pharmaceutically acceptable solvate thereof.
 2. The methodaccording to claim 1, wherein the method is for treating a disease, andthe disease is Parkinson's disease.
 3. The method according to claim 1,wherein the method is for delaying the onset of a disease, and thedisease is Alzheimer's disease.
 4. The method according to claim 1wherein the sulphate salt has Formula II

wherein x designates the number of equivalents of sulphuric acid and xis 0.5 to 4, or a pharmaceutically acceptable solvate thereof.
 5. Themethod according to claim 2 wherein the sulphate salt has Formula II

wherein x designates the number of equivalents of sulphuric acid and xis 0.5 to 4, or a pharmaceutically acceptable solvate thereof.
 6. Themethod according to claim 3 wherein the sulphate salt has Formula II

wherein x designates the number of equivalents of sulphuric acid and xis 0.5 to 4, or a pharmaceutically acceptable solvate thereof.
 7. Amethod according to claim 1 wherein the method is for treating adisease, and the disease is Alzheimer's disease.
 8. A method accordingto claim 1 wherein the method is for delaying the onset of a disease,and the disease is Parkinson's disease.
 9. The method according to claim7 wherein the sulphate salt has Formula II

wherein x designates the number of equivalents of sulphuric acid and xis 0.5 to 4, or a pharmaceutically acceptable solvate thereof.
 10. Themethod according to claim 8 wherein the sulphate salt has Formula II

wherein x designates the number of equivalents of sulphuric acid and xis 0.5 to 4, or a pharmaceutically acceptable solvate thereof.